Glycobiology Advance Access originally published online on July 8, 2003
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Glycobiology, 2003, Vol. 13, No. 10 693-706
© 2003 Oxford University Press
Topological characterization of an inner membrane (1
3)-ß-D-glucan (curdlan) synthase from Agrobacterium sp. strain ATCC31749
2 Department of Biochemistry, La Trobe University, Melbourne, Victoria 3086, Australia; 3 CSIRO Division of Health Sciences and Nutrition, 343 Royal Parade, Parkville, Victoria 3052, Australia; and 4 Department of Microbiology, La Trobe University, Melbourne, Victoria 3086, Australia
Received on April 13, 2003; revised on June 5, 2003; accepted on June 10, 2003
The crdS gene of Agrobacterium sp. strain ATCC31749 encodes the curdlan synthase (CrdS) protein based on the homology of the derived CrdS protein sequence with those of ß-glycosyl transferases with repetitive action patterns (Stasinopoulos et al. [1999] Glycobiology, 9, 31–41). Here we show that chemical (NTG) mutagenesis of crdS abolishes curdlan production and the induced mutations can be complemented by a cloned crdS amplicon, thus providing genetic confirmation that crdS is essential for curdlan production. When expressed in the native Agrobacterium or in Escherichia coli, the largely hydrophobic CrdS protein exhibited an Mr of 
60 kDa (compared with the predicted mass of 73,121 Da) and was located in the inner membrane of both bacteria. By analyzing reciprocal fusions between crdS and the reporter genes, lacZ and phoA, and assessing the sensitivity of CrdS in spheroplasts to proteinase K, CrdS was shown to be an integral membrane protein with seven transmembrane helices and an Nout–Cin disposition. A central large and relatively hydrophilic cytoplasmic region carries the substrate-binding and catalytic D,D,D35QxxRW motif. The amino acid sequence of this domain of CrdS was threaded onto the 3D structure of the comparable domain of the SpsA protein, a member of the family GT-2 glycosyl transferases, and enabled the identification of corresponding amino acids involved in binding UDP in CrdS. Analysis of Agrobacterium membrane preparations using blue native–PAGE provided preliminary evidence that CrdS occurs in multimeric protein complexes of 420 kDa and 500 kDa.
1 To whom correspondence should be addressed; email: v.stanisich{at}latrobe.edu.au
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